The present invention is directed generally to a device that combines a miniaturizable high pressure pumping means and nozzle for generating a high pressure spray and particularly to a device for injecting DNA, chemotherapeutic agents, and vaccines into cells and tissues. Because of its small size the device can be incorporated into an endoscope or catheter, thereby providing non-invasive access to difficult to reach tissues, such as intestinal epithelium or the left ventricle interior wall, for therapy.
There is, at present, a great deal of interest in the medical technologies and biotechnology in applications that allow the insertion of genes into live eukaryotic cells and tissues. Two new areas of medical research, gene therapy, and DNA vaccines, that may have revolutionary impact of the practice of medicine, depend on such gene insertion techniques.
A surprisingly successful method for the introduction of DNA into cells has been the physical bombardment of cells and tissues with high-speed droplets or particles carrying DNA as discussed in Yang, N. S., and Sun, W. H., Nature Medicine, 1, 481-483, 1995; Johnston, S. A. and Tang, D. C., Methods in Cell Biology, 43, 353-365, 1994; Fynan, E. F. et al., Proceedings of the National Academy of Sciences of the United States of America, 11478-11482, 1993. In the first implementation of this technique (Sanford, J. C. et al., Part. Sci, Technol., 5, 27-37, 1987) the DNA was literally shot through the walls of the target cells. Although initially developed for use in plant cell lines, this approach has been found to result in significant levels of incorporation and expression of the DNA in a wide range of targets, including mammalian cells in culture, intact rat liver, and skin. The success of the strategy is surprising, not in the least, because it requires that cells remain viable after having submicron-size holes torn in their membranes, although it is not clear whether DNA must be ballistically transported into the cells, or simply the cells permeabilized to allow DNA on or around the cells to diffuse or be transported in. Other (seemingly more controlled) techniques for introducing DNA that are successful in vitro, such as single-cell microinjection, and electroporation, are not easily adaptable for clinical in situ gene therapy. Techniques that rely on vectors such as viruses or liposomes to deliver DNA to target tissues have other limitations, such as the need to find a vector that will target a certain tissue or cell type and will be specific to that cell type. If such vectors are injected, the region of transfected cells is not spatially localized (except by means of biological specificity). Bombardment methods have the advantage that they allow DNA to be introduced without the need for packing it in a vector, and the DNA can then be aimed (quite literally) at accessible target cells, though it is also possible to shoot viral particles into cells.
One effective means for DNA bombardment of cells is the use of a shock tube. In one example of such an instrument (Nabuisi, S. M., et al., Measurement Sci. and Tech., 5, 267-274, 1994), a membrane, located at the entrance of the tube, is ruptured by a high pressure gas to create a shock wave that impinges upon a secondary membrane at the other end of the tube. This secondary membrane is coated with DNA-containing particles, such as DNA adsorbed onto gold beads. When the shock wave hits it, the particles are accelerated to supersonic velocities and embed themselves in the target cells or tissues. Another simpler method is the use of a traditional jet injector (Haensler, J., et al., Vaccine, 17, 628-638, 1999), which consists in its most basic form of a spring-loaded plunger in a cylinder barrel with a small-aperture nozzle. When the plunger is suddenly released, the cylinder acts as a hydraulic intensifier, and a high pressure (e.g., 10 MPa) stream is emitted from the nozzle with velocities on the order of 140 m/sec. There is an apparent predominance of shock-tube or similar gas-driven methods in the scientific literature, probably related to their demonstrated efficacy for a variety of applications. This may in turn be due to the higher particle velocities that are achieved, allowing more cells to be permeated and deeper penetration into tissues and easier delivery of DNA through liquid layers and dead cells.
There is a long history of the use of jet injectors for intra/subcutaneous vaccinations (Hingston, R. A. and Hughes, J. G., Anesth. Analg. Cleve., 221-242, 1947). They are familiar, in some variant, to much of the population from their use in mass immunization programs. Shock-tube-based methods may also be used on skin as the target organ, or on surgically exposed tissues, as mentioned above. However, it is not straightforward to create a shock tube or high pressure gas feed system that can be safely used inside a body cavity or the cardiovascular system, and this factor limits the applicability of these strategies for introducing DNA into diseased tissues. Aside from the obvious pressure safety concerns, shock-tube methods would be precluded in the cardiovascular system, where any gas release would be unacceptable. More fundamentally, miniaturization of jet and shock-based designs is problematical because of simple hydrodynamic scaling issues. For example, to maximize nozzle velocities of jet injections, is it desirable to have a large diameter cylinders in order to minimize 0-ring frictional losses and pressure drop in the barrel. For constant nozzle velocity, these losses increase as the inverse square of the barrel radius, and gas flows in small diameter tubes have similar problems.
Consideration of the above discussion leads to several design features that should be met by a miniaturized DNA delivery device for internal use, in addition to demonstrated efficacy for performing DNA transfer:
1) The principle of operation should be such that performance does not deteriorate as the device is miniaturized, and the device should fit in the endoscope lumen (approximately 0.5 cm in diameter).
2) No high pressure gas supply lines that would have to run along the endoscope.
3) Only physiologically compatible fluids or particles released, with no release of gases.
4) Injection velocities should be substantially higher than that of jet injectors.
5) Power must be supplied by small wires that could be fit into an endoscope or catheter.
6) The device must be sterilizable.
7) The device should allow arrays of nozzles to be used.
8) The device should be able to be manufactured as a disposable item.
The present invention makes use of miniature high pressure pump technology combined with a nozzle having a sub-micron aperture or orifice to provide a novel device for delivering high pressure sprays at low flow rates, generally, and for direct gene/DNA delivery into cells that is suitable for clinical use on the end of an endoscope or catheter, in particular. Thus, in contrast to prior art devices for introduction of DNA into cells, the present device provides for direct genetic manipulation of difficult to access tissues, such as intestinal epithelium or the left ventricular wall, through relatively non-invasive means and thus, enhances the ability to address diseases of major importance such as heart disease and cancer of the alimentary tract. Further, the novel device disclosed herein is also generally useful for microinjection of chemo-therapeutic agents or vaccines into skin and other tissue. Furthermore, the inventive device has no moving parts, is constructed of simple materials, is compatible with micromachining such that inexpensive mass production is possible, releases only very small amounts of gas, provides injection velocities substantially higher than prior art devices such as jet injectors, and is sterilizable.
Generally, the inventive device integrates a high pressure electrokinetic pump (EKP) with a micro-nozzle, i.e., a nozzle having an aperture or orifice whose opening is sub-micron in diameter, for the delivery of a high pressure, low flow rate spray, including delivery of DNA or other biological materials into tissues. It has been demonstrated that the EKP is capable of providing levels of very high pressure ( greater than 104 psi or 100 Mpa) at low flow rates. Thus, the present device can evolve higher pressures than prior art spring-actuated devices or even shock tubes. Moreover, by using an EKP for pressure generation, the need for high pressure lines is obviated and thus the pressure losses that are encountered in prior art devices that transport hydraulic power from the pressure generator to the point of use are eliminated.